Bachelor of Science, University of Richmond (2009)
Doctor of Philosophy, S.U.N.Y. State University at Stony Brook (2015)
Maria Grazia Roncarolo, Postdoctoral Faculty Sponsor
View details for Web of Science ID 000464381005080
The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.
View details for PubMedID 30692695
Herpesviruses establish a chronic infection in the host characterized by intervals of lytic replication, quiescent latency, and reactivation from latency. Murine gammaherpesvirus 68 (MHV68) naturally infects small rodents and has genetic and biologic parallels with the human gammaherpesviruses (gHVs), Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. The murine gammaherpesvirus model pathogen system provides a platform to apply cutting-edge approaches to dissect the interplay of gammaherpesvirus and host determinants that enable colonization of the host, and that shape the latent or lytic fate of an infected cell. This knowledge is critical for the development of novel therapeutic interventions against the oncogenic gHVs. The nuclear factor kappa B (NF-?B) signaling pathway is well-known for its role in the promotion of inflammation and many aspects of B cell biology. Here, we review key aspects of the virus lifecycle in the host, with an emphasis on the route that the virus takes to gain access to the B cell latency reservoir. We highlight how the murine gammaherpesvirus requires components of the NF-?B signaling pathway to promote replication, latency establishment, and maintenance of latency. These studies emphasize the complexity of gammaherpesvirus interactions with NF-?B signaling components that direct innate and adaptive immune responses of the host. Importantly, multiple facets of NF-?B signaling have been identified that might be targeted to reduce the burden of gammaherpesvirus-associated diseases.
View details for DOI 10.3389/fmicb.2016.01202
View details for Web of Science ID 000381548900001
View details for PubMedID 27582728
Gammaherpesviruses establish lifelong infections that are associated with the development of cancer. These viruses subvert many aspects of the innate and adaptive immune response of the host. The inflammasome, a macromolecular protein complex that controls inflammatory responses to intracellular danger signals generated by pathogens, is both activated and subverted during human gammaherpesvirus infection in culture. The impact of the inflammasome response on gammaherpesvirus replication and latency in vivo is not known. Caspase-1 is the inflammasome effector protease that cleaves the proinflammatory cytokines interleukin-1? (IL-1?) and IL-18. We infected caspase-1-deficient mice with murine gammaherpesvirus 68 (MHV68) and observed no impact on acute replication in the lung or latency and reactivation from latency in the spleen. This led us to examine the effect of viral infection on inflammasome responses in bone marrow-derived macrophages. We determined that infection of macrophages with MHV68 led to a robust interferon response but failed to activate caspase-1 or induce the secretion of IL-1?. In addition, MHV68 infection led to a reduction in IL-1? production after extrinsic lipopolysaccharide stimulation or upon coinfection with Salmonella enterica serovar Typhimurium. Interestingly, this impairment occurred at the proIL-1? transcript level and was independent of the RTA, the viral lytic replication and transcription activator. Taken together, MHV68 impairs the inflammasome response by inhibiting IL-1? production during the initial stages of infection.Gammaherpesviruses persist for the lifetime of the host. To accomplish this, they must evade recognition and clearance by the immune system. The inflammasome consists of proteins that detect foreign molecules in the cell and respond by secreting proinflammatory signaling proteins that recruit immune cells to clear the infection. Unexpectedly, we found that murine gammaherpesvirus pathogenesis was not enhanced in mice lacking caspase-1, a critical inflammasome component. This led us to investigate whether the virus actively impairs the inflammasome response. We found that the inflammasome was not activated upon macrophage cell infection with murine gammaherpesvirus 68. Infection also prevented the host cell inflammasome response to other pathogen-associated molecular patterns, indicated by reduced production of the proinflammatory cytokine IL-1? upon bacterial coinfection. Taken together, murine gammaherpesvirus impairment of the inflammatory cytokine IL-1? in macrophages identifies one mechanism by which the virus may inhibit caspase-1-dependent immune responses in the infected animal.
View details for DOI 10.1128/JVI.00658-15
View details for Web of Science ID 000355593000005
View details for PubMedID 25855746
The human gammaherpesviruses establish life-long infections that are associated with the development of lymphomas and neoplasms, especially in immunocompromised individuals. T cells play a crucial role in the control of gammaherpesvirus infection through multiple functions, including the direct killing of infected cells, production of cytokines such as interferon-? (IFN-?), and costimulation of B cells. Impaired T cell function in mice infected with murine gammaherpesvirus 68 (MHV68) leads to increased reactivation and pathologies, including a higher incidence of lymphoid hyperplasia. Here we report that the absence of Suppressor of TCR signaling -1 and -2 (Sts-1(-/-)/2(-/-)) during MHV68 infection leads to the generation of T cells with significantly heightened responses. Transient differences in the T and B cell response of infected Sts-1(-/-)/2(-/-) (Sts dKO) mice were also observed when compared to WT mice. However, these alterations in the immune response and the overall absence of Sts-1 and Sts-2 did not impact viral pathogenesis or lead to pathology. Acute lytic replication in the lungs, establishment of latency in the spleen and reactivation from latency in the spleen in the Sts dKO mice were comparable to WT mice. Our studies indicate that Sts-1 and Sts-2 are not required for the immune control of MHV68 in a normal course of gammaherpesvirus infection, but suggest that interference with negative regulators of T cell responses might be further explored as a safe and efficacious strategy to improve adoptive T cell therapy.
View details for DOI 10.1371/journal.pone.0090196
View details for Web of Science ID 000332396200146
View details for PubMedID 24587276
Examining the origins of highly conserved gene regulatory networks (GRNs) will inform our understanding of the evolution of animal body plans. Sponges are believed to be the most ancient extant metazoan lineage, and as such, hold clues about the evolution of genetic programs deployed in animal development. We used the emerging freshwater sponge model, Ephydatia muelleri, to study the evolutionary origins of the Pax/Six/Eya/Dac (PSED) GRN. Orthologs to Pax and Six family members are present in E. muelleri and are expressed in endothelial cells lining the canal system as well as cells in the choanoderm. Knockdown of EmPaxB and EmSix1/2 by RNAi resulted in defects to the canal systems. We further show that PaxB may be in a regulatory relationship with Six1/2 in E. muelleri, thus demonstrating that a component of the PSED network was present early in metazoan evolution.
View details for DOI 10.1111/ede.12032
View details for Web of Science ID 000318229300003
View details for PubMedID 23607302
The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available.We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene.This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.
View details for DOI 10.1186/1472-6750-11-67
View details for Web of Science ID 000293267400001
View details for PubMedID 21679422
View details for PubMedCentralID PMC3146823